A direct spino-cortical circuit bypassing the thalamus modulates nociception.

Nociceptive signals are usually transmitted to layer 4 neurons in somatosensory cortex via the spinothalamic-thalamocortical pathway. The layer 5 corticospinal neurons in sensorimotor cortex are reported to receive the output of neurons in superficial layers; and their descending axons innervate the spinal cord to regulate basic sensorimotor functions. Here, we show that a subset of layer 5 neurons receives spinal inputs through a direct spino-cortical circuit bypassing the thalamus, and thus define these neurons as spino-cortical recipient neurons (SCRNs). Morphological studies revealed that the branches from spinal ascending axons formed a kind of disciform structure with the descending axons from SCRNs in the basilar pontine nucleus (BPN). Electron microscopy and calcium imaging further confirmed that the axon terminals from spinal ascending neurons and SCRNs made functional synaptic contacts in the BPN, linking the ascending sensory pathway to the descending motor control pathway. Furthermore, behavioral tests indicated that the spino-cortical connection in the BPN was involved in nociceptive responses. In vivo calcium imaging showed that SCRNs responded to peripheral noxious stimuli faster than neighboring layer 4 cortical neurons in awake mice. Manipulating activities of SCRNs could modulate nociceptive behaviors. Therefore, this direct spino-cortical circuit represents a noncanonical pathway, allowing a fast sensory-motor transition of the brain in response to noxious stimuli.

Distinct neural networks derived from galanin-containing nociceptors and neurotensin-expressing pruriceptors.

Pain and itch are distinct sensations arousing evasion and compulsive desire for scratching, respectively. It's unclear whether they could invoke different neural networks in the brain. Here, we use the type 1 herpes simplex virus H129 strain to trace the neural networks derived from two types of dorsal root ganglia (DRG) neurons: one kind of polymodal nociceptors containing galanin (Gal) and one type of pruriceptors expressing neurotensin (Nts). The DRG microinjection and immunosuppression were performed in transgenic mice to achieve a successful tracing from specific types of DRG neurons to the primary sensory cortex. About one-third of nuclei in the brain were labeled. More than half of them were differentially labeled in two networks. For the ascending pathways, the spinothalamic tract was absent in the network derived from Nts-expressing pruriceptors, and the two networks shared the spinobulbar projections but occupied different subnuclei. As to the motor systems, more neurons in the primary motor cortex and red nucleus of the somatic motor system participated in the Gal-containing nociceptor-derived network, while more neurons in the nucleus of the solitary tract (NST) and the dorsal motor nucleus of vagus nerve (DMX) of the emotional motor system was found in the Nts-expressing pruriceptor-derived network. Functional validation of differentially labeled nuclei by c-Fos test and chemogenetic inhibition suggested the red nucleus in facilitating the response to noxious heat and the NST/DMX in regulating the histamine-induced scratching. Thus, we reveal the organization of neural networks in a DRG neuron type-dependent manner for processing pain and itch.

Zcchc12-Containing Nociceptors Are Required for Noxious Heat Sensation.

DRG neurons are classified into distinct types to mediate the somatosensation with different modalities. Recently, transcriptional profilings of DRG neurons by single-cell RNA-sequencing have provided new insights into the neuron typing and functional properties. Zinc-finger CCHC domain-containing 12 (Zcchc12) was reported to be the representative marker for a subtype of galanin-positive (Gal+) DRG neurons. However, the characteristics and functions of Zcchc12+ neurons are largely unknown. Here, we genetically labeled Zcchc12+ neurons in Zcchc12-CreERT2::Ai9 mice, and verified that Zcchc12 represented a new subpopulation of DRG neurons in both sexes. Zcchc12+ neurons centrally innervated the superficial laminae in spinal dorsal horn, and peripherally terminated as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. In addition, Zcchc12+ neurons also formed circumferential endings surrounding the hair follicles in hairy skin. Functionally, in vivo calcium imaging in DRGs revealed that Zcchc12+ neurons were polymodal nociceptors and could be activated by mechanical and noxious thermal stimuli. Behavioral tests showed that selective ablation of Zcchc12+ DRG neurons reduced the sensitivity to noxious heat in mice. Together, we identified a new subpopulation of Zcchc12+ nociceptors essential for noxious heat sensation.SIGNIFICANCE STATEMENTZcchc12 represents a new subpopulation of DRG neurons. The characteristics and functions of Zcchc12+ neurons are largely unknown. Here we genetically labeled Zcchc12+ neurons, and showed that the fibers of Zcchc12+ DRG neurons projected to superficial lamina at spinal dorsal horn, and innervated skin as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Functionally, Zcchc12+ DRG neurons responded to noxious mechanical and heat stimuli. Ablation of Zcchc12+ DRG neurons impaired the sensation of noxious heat in mice. Therefore, we identify a new subpopulation of DRG neurons required for noxious heat sensation.

5'-UTR SNP of FGF13 causes translational defect and intellectual disability.

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5'-untranslated region (5'-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5'-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5'-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5'-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.

Single-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic pain.

Peripheral nerve injury could lead to chronic neuropathic pain. Understanding transcriptional changes induced by nerve injury could provide fundamental insights into the complex pathogenesis of neuropathic pain. Gene expression profiles of dorsal root ganglia (DRG) in neuropathic pain condition have been studied. However, little is known about transcriptomic changes in individual DRG neurons after peripheral nerve injury. Here we performed single-cell RNA sequencing on dissociated mouse DRG cells after spared nerve injury (SNI). In addition to DRG neuron types that are found under physiological conditions, we identified three SNI-induced neuronal clusters (SNIICs) characterized by the expression of Atf3/Gfra3/Gal (SNIIC1), Atf3/Mrgprd (SNIIC2) and Atf3/S100b/Gal (SNIIC3). These SNIICs originated from Cldn9+/Gal+, Mrgprd+ and Trappc3l+ DRG neurons, respectively. Interestingly, SNIIC2 switched to SNIIC1 by increasing Gal and reducing Mrgprd expression 2 days after nerve injury. Inferring the gene regulatory networks after nerve injury, we revealed that activated transcription factors Atf3 and Egr1 in SNIICs could enhance Gal expression while activated Cpeb1 in SNIIC2 might suppress Mrgprd expression within 2 days after SNI. Furthermore, we mined the transcriptomic changes in the development of neuropathic pain to identify potential analgesic targets. We revealed that cardiotrophin-like cytokine factor 1, which activates astrocytes in the dorsal horn of spinal cord, was upregulated in SNIIC1 neurons and contributed to SNI-induced mechanical allodynia. Therefore, our results provide a new landscape to understand the dynamic course of neuron type changes and their underlying molecular mechanisms during the development of neuropathic pain.

Nerve Injury-Induced Neuronal PAP-I Maintains Neuropathic Pain by Activating Spinal Microglia.

Pancreatitis-associated proteins (PAPs) display multiple functions in visceral diseases. Previous studies showed that the expression level of PAP-I was low in the DRG of naive rats but was de novo expressed after peripheral nerve injury. However, its role in neuropathic pain remains unknown. We found that PAP-I expression was continuously upregulated in the DRG neurons from rat spared nerve injury models, and transported toward the spinal dorsal horn to act as a proinflammatory factor. Intrathecal delivery of PAP-I enhanced sensory hyperalgesia, whereas PAP-I deficiency by either gene knockout or antibody application alleviated tactile allodynia at the maintenance phase after spared nerve injury. Furthermore, PAP-I functioned by activating the spinal microglia via C-C chemokine receptor Type 2 that participated in neuropathic pain. Inhibition of either microglial activation or C-C chemokine receptor Type 2 abolished the PAP-I-induced hyperalgesia. Thus, PAP-I mediates the neuron-microglial crosstalk after peripheral nerve injury and contributes to the maintenance of neuropathic pain.SIGNIFICANCE STATEMENT Neuropathic pain is maladaptive pain condition, and the maintaining mechanism is largely unclear. Here we reveal that, after peripheral nerve injury, PAP-I can be transported to the spinal dorsal horn and is crucial in the progression of neuropathic pain. Importantly, we prove that PAP-I mainly functions through activating the spinal microglia via the CCR2-p38 MAPK pathway. Furthermore, we confirm that the proinflammatory effect of PAP-I is more prominent after the establishment of neuropathic pain, thus indicating that microglia also participate in the maintenance phase of neuropathic pain.

Clinical opioids differentially induce co-internalization of µ- and δ-opioid receptors.

Opioid receptors play an important role in mediating the spinal analgesia. The µ-opioid receptor is the major target of opioid drugs widely used in clinics. However, the regulatory mechanisms of analgesic effect and tolerance for clinical µ-opioid receptor-targeting opioids remain to be fully investigated. Previous studies showed the interaction of δ-opioid receptor with µ-opioid receptor to form the µ-opioid receptor/δ-opioid receptor heteromers that could be processed in the degradation pathway after δ-opioid receptor agonist treatment. Here, we showed that clinical µ-opioid receptor-targeting opioids, morphine, fentanyl, and methadone, but not tramadol, caused µ-opioid receptor co-internalization with δ-opioid receptors in both transfected human embryonic kidney 293 cells and primary sensory neurons. Prolonged treatment of morphine led to µ-opioid receptor co-degradation with δ-opioid receptors. Furthermore, fentanyl and methadone, but not tramadol, induced the drug tolerance similar to morphine. Thus, the clinical µ-opioid receptor-targeting opioids including morphine, fentanyl, and methadone induce µ-opioid receptor co-internalization with δ-opioid receptors, which may be involved in the analgesic tolerance of these opioids.

FGF13 Selectively Regulates Heat Nociception by Interacting with Nav1.7.

The current knowledge about heat nociception is mainly confined to the thermosensors, including the transient receptor potential cation channel V1 expressed in the nociceptive neurons of dorsal root ganglion (DRG). However, the loss of thermosensors only partially impairs heat nociception, suggesting the existence of undiscovered mechanisms. We found that the loss of an intracellular fibroblast growth factor (FGF), FGF13, in the mouse DRG neurons selectively abolished heat nociception. The noxious heat stimuli could not evoke the sustained action potential firing in FGF13-deficient DRG neurons. Furthermore, FGF13 interacted with the sodium channel Nav1.7 in a heat-facilitated manner. FGF13 increased Nav1.7 sodium currents and maintained the membrane localization of Nav1.7 during noxious heat stimulation, enabling the sustained firing of action potentials. Disrupting the FGF13/Nav1.7 interaction reduced the heat-evoked action potential firing and nociceptive behavior. Thus, beyond the thermosensors, the FGF13/Nav1.7 complex is essential for sustaining the transmission of noxious heat signals.

[Development of three-dimensional breast cancer cell culture drug resistance model].

The aim of the present study was to develop three-dimensional (3D) culture model, a more pathologically relevant model, of human breast cancer for drug resistance study. MCF-7 cells were embedded within collagen gel to establish 3D culture model. Cellular morphology was observed using Carmine and HE staining. Cell proliferation was evaluated by CCK-8 assay, and cell activity was detected by Live/Dead staining kit. Drug sensitivities of the 3D culture to doxorubicin, carboplatin, 5-fluorouracil were assayed and compared with those of monolayer (2D) culture. In addition, the levels of drug resistance-related genes P-glycoprotein (P-gp), mrp2 mRNA expressions were detected by real time RT-PCR. Expression level of P-gp protein was detected by Western blot. The results showed that MCF-7 cells in 3D culture formed a number of cell aggregates, and most of them displayed good cell viability. The IC50 values of doxorubicin, carboplatin, 5-fluorouracil were all increased significantly in 3D culture compared with those in 2D culture. Moreover, compared with MCF-7 cells in 2D culture, the cells in 3D culture showed increased mRNA levels of P-gp and mrp2, as well as up-regulated protein expression of P-gp. These results suggest that in vitro collagen-embedded culture system of human breast cancer cells represents an improved pathologically relevant 3D microenvironment for breast cancer cells, providing a robust tool to explore the mechanism of drug resistance of cancer cells.

Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity.

Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion (DRG) by high-coverage single-cell RNA-sequencing (10 950 ± 1 218 genes per neuron) and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors (MHNs). Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors (MNs) expressing BAI1-associated protein 2-like 1 (Baiap2l1). Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap2l1-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases.

FXYD2, a γ subunit of Na⁺, K⁺-ATPase, maintains persistent mechanical allodynia induced by inflammation.

Na⁺, K⁺-ATPase (NKA) is required to generate the resting membrane potential in neurons. Nociceptive afferent neurons express not only the α and ß subunits of NKA but also the γ subunit FXYD2. However, the neural function of FXYD2 is unknown. The present study shows that FXYD2 in nociceptive neurons is necessary for maintaining the mechanical allodynia induced by peripheral inflammation. FXYD2 interacted with α1NKA and negatively regulated the NKA activity, depolarizing the membrane potential of nociceptive neurons. Mechanical allodynia initiated in FXYD2-deficient mice was abolished 4 days after inflammation, whereas it persisted for at least 3 weeks in wild-type mice. Importantly, the FXYD2/α1NKA interaction gradually increased after inflammation and peaked on day 4 post inflammation, resulting in reduction of NKA activity, depolarization of neuron membrane and facilitation of excitatory afferent neurotransmission. Thus, the increased FXYD2 activity may be a fundamental mechanism underlying the persistent hypersensitivity to pain induced by inflammation.

Activin C expressed in nociceptive afferent neurons is required for suppressing inflammatory pain.

Emerging evidence suggests that the suppressive modulators released from nociceptive afferent neurons contribute to pain regulation. However, the suppressive modulators expressed in small-diameter neurons of the dorsal root ganglion remain to be further identified. The present study shows that the activin C expressed in small dorsal root ganglion neurons is required for suppressing inflammation-induced nociceptive responses. The expression of activin C in small dorsal root ganglion neurons of rats was markedly downregulated during the early days of peripheral inflammation induced by intraplantar injection of the complete Freund's adjuvant. Intrathecal treatment with the small interfering RNA targeting activin ßC or the antibodies against activin C could enhance the formalin-induced nociceptive responses, and impair the recovery from the complete Freund's adjuvant-induced thermal hyperalgesia. Intrathecally applied activin C could reduce nociceptive responses induced by formalin or complete Freund's adjuvant. Moreover, activin C was found to inhibit the inflammation-induced phosphorylation of extracellular signal-regulated kinase in the dorsal root ganglia and the dorsal spinal cord. Thus, activin C functions as an endogenous suppressor of inflammatory nociceptive transmission and may have a therapeutic potential for treatment of inflammatory pain.

Follistatin-like 1 suppresses sensory afferent transmission by activating Na+,K+-ATPase.

Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.

Transport of receptors, receptor signaling complexes and ion channels via neuropeptide-secretory vesicles.

Stimulus-induced exocytosis of large dense-core vesicles (LDCVs) leads to discharge of neuropeptides and fusion of LDCV membranes with the plasma membrane. However, the contribution of LDCVs to the properties of the neuronal membrane remains largely unclear. The present study found that LDCVs were associated with multiple receptors, channels and signaling molecules, suggesting that neuronal sensitivity is modulated by an LDCV-mediated mechanism. Liquid chromatography-mass spectrometry combined with immunoblotting of subcellular fractions identified 298 proteins in LDCV membranes purified from the dorsal spinal cord, including G-protein-coupled receptors, G-proteins and other signaling molecules, ion channels and trafficking-related proteins. Morphological assays showed that δ-opioid receptor 1 (DOR1), ß2 adrenergic receptor (AR), G(αi2), voltage-gated calcium channel α2δ1 subunit and P2X purinoceptor 2 were localized in substance P (SP)-positive LDCVs in small-diameter dorsal root ganglion neurons, whereas ß1 AR, Wnt receptor frizzled 8 and dishevelled 1 were present in SP-negative LDCVs. Furthermore, DOR1/G(αi2)/G(ß1γ5)/phospholipase C ß2 complexes were associated with LDCVs. Blockade of the DOR1/G(αi2) interaction largely abolished the LDCV localization of G(αi2) and impaired stimulation-induced surface expression of G(αi2). Thus, LDCVs serve as carriers of receptors, ion channels and preassembled receptor signaling complexes, enabling a rapid, activity-dependent modulation of neuronal sensitivity.

Facilitation of µ-opioid receptor activity by preventing δ-opioid receptor-mediated codegradation.

δ-opioid receptors (DORs) form heteromers with µ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR (MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane is a potential strategy to improve opioid analgesic therapies.

Inhibition of inflammatory pain by activating B-type natriuretic peptide signal pathway in nociceptive sensory neurons.

B-type natriuretic peptide (BNP) has been known to be secreted from cardiac myocytes and activate its receptor, natriuretic peptide receptor-A (NPR-A), to reduce ventricular fibrosis. However, the function of BNP/NPR-A pathway in the somatic sensory system has been unknown. In the present study, we report a novel function of BNP in pain modulation. Using microarray and immunoblot analyses, we found that BNP and NPR-A were expressed in the dorsal root ganglion (DRG) of rats and upregulated after intraplantar injection of complete Freund's adjuvant (CFA). Immunohistochemistry showed that BNP was expressed in calcitonin gene-related peptide (CGRP)-containing small neurons and IB4 (isolectin B4)-positive neurons, whereas NPR-A was present in CGRP-containing neurons. Application of BNP reduced the firing frequency of small DRG neurons in the presence of glutamate through opening large-conductance Ca2+-activated K+ channels (BKCa channels). Furthermore, intrathecal injection of BNP yielded inhibitory effects on formalin-induced flinching behavior and CFA-induced thermal hyperalgesia in rats. Blockade of BNP signaling by BNP antibodies or cGMP-dependent protein kinase (PKG) inhibitor KT5823 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester] impaired the recovery from CFA-induced thermal hyperalgesia. Thus, BNP negatively regulates nociceptive transmission through presynaptic receptor NPR-A, and activation of the BNP/NPR-A/PKG/BKCa channel pathway in nociceptive afferent neurons could be a potential strategy for inflammatory pain therapy.

Peripheral nerve injury induces reorganization of galanin-containing afferents in the superficial dorsal horn of monkey spinal cord.

Peripheral nerve injury-induced structural and chemical modifications of the sensory circuits in the dorsal horn of the spinal cord contribute to the mechanism of neuropathic pain. In contrast to the topographic projection of primary afferents in laminae I-IV in the rat spinal cord, the primary afferents of Macaca mulatta monkeys almost exclusively project into laminae I-II of the spinal cord. After peripheral nerve injury, up-regulation of galanin has been found in sensory neurons in both monkey and rat dorsal root ganglia. However, the nerve injury-induced ultrastructural modification of galanin-containing afferents in the monkey spinal cord remains unknown. Using immunoelectron microscopy, we found that 3 weeks after unilateral sciatic nerve transection, the number of galanin-containing afferents was increased in ipsilateral lamina II of monkey spinal cord. Branching of these galanin-positive afferents was often observed. The afferent terminals contained a large number of synaptic vesicles, peptidergic vesicles and mitochondria, whereas the number of synapses was markedly reduced. Some of the afferents-enriched microtubules were often packed into bundles. Moreover, galanin-labeling could be associated with endosomal structures in many dendrites and axonal terminals of dorsal horn neurons. These results suggest that peripheral nerve injury induces an expansion of the central projection of galanin-containing afferents in lamina II of the monkey spinal cord, not only by increasing galanin levels in primary afferents but also by triggering afferent branching.